Process for (A) separating biological/ligands from dilute solutions and (B) conducting an immunochromatographic assay thereof employing superparamagnetic particles throughout

ABSTRACT

Superparamagnetic (“SPM”) subunits of 1-30 nm average mean diameter (e.g. ferro fluid) subparticles are treated with a magnetically noninterfering substance capable of coating and covering them (e.g, BSA) and they spontaneously form agglomerates of about 100 nm to about 450 nm or higher average mean diameter and are then used to form complexes with target biological ligands such as viruses, contained in large volumes of liquid. The complexes are subjected to the gradient intensity of a strong magnetic field, and excess liquid is removed, where upon an immunochromatographic assay is conducted to determine the identity and/or amount of target ligand present, in which operation SPM particles that bonded to the ligand function as tags for ligand detection.

This application is a continuation of U.S. patent application Ser. No.10/044,920 filed Jan. 15, 2002, now U.S. Pat. No. ______. This inventionrelates to using the same superparamagnetic particles, as moreparticularly described hereinafter, to concentrate biological substancesbelieved to be sparsely present in large volumes of fluids and aslabelling agents for detecting the quantity of the same biologicalmolecules present in a fluid sample.

BACKGROUND OR THE INVENTION

Heretofore it has become common to use metallic particles havingsuperparamagnetic properties to concentrate biological ligands presentin small amounts in large volumes of aqueous fluids, including fluids ofmammalian origin such as urine. These metallic particles are often oflarge size (typically in the order of 1-5 μm or larger in average meandiameter) such that they cannot move, or do not move sufficientlyreadily, through the matrices used for either flow-through tests orlateral flow immunochromatographic (“ICT”) tests such as those commonlyused currently in many commercially available diagnostic tests foridentifying disease causative pathogens. In an instance where such testsare to follow the initial concentration step, removal of thesuperparamagnetic particles used for concentration is necessary,followed by adding a target specific conjugate labelled with achemiluminescent, fluorescent or radioactive tag, or a tag such ascolloidal latex particles, colloidal gold, or another colloidal metalwhich couples to the biological ligand and aids in the detectionthereof. The need to remove superparamagnetic particles used in ligandconcentration and then subject the concentrated ligand to anidentification or quantification assay often poses problems. Forexample, quantification of the small amount of biological ligandobtained by concentration is rendered inaccurate if even a tiny fragmentof concentrated ligand clings to the particles used for concentration;by the same token, incomplete removal of a small fragment of a magneticparticle may disrupt a qualitative identification of the concentratedsample by setting up an interaction with the labelling agent chosen foruse in the subsequent identification test. Even in cases wheresuperparamagnetic particles are employed to concentrate biologicalligands present in a large volumes of fluid and the nature of thesubsequent identification procedure renders separation of thesuperparamagnetic particles unnecessary, these particles haveheretofore-been viewed in the art as irrelevant to the subsequentidentification step.

Large sized superparamagnetic particles have been preferred for ligandconcentration work, because their large size (in the order of 1 to 5 μmor more) increases the mass of material bound to the target ligand andallows the gradient field of a fixed magnet to effect separation withease. Much smaller particles have been used in some instances but oftenthe low mass of magnetic material that they impart to their target,requires the introduction of magnetizable columns, filters or screens asan aid to separating the target molecules from the sample.

Particles heretofore used as tags for detecting a biological ligand(regardless of whether it has been subjected to a first concentrationstep) are usually quite small. As already noted, this is especially truewhere rapid “flow-through” or lateral flow matrices having narrow poresare employed as solid phase substrates. Particularly in the lateral flowICT format, particles used as detection markers must be small enough tomigrate through the pores of the matrices and reach the immobilizedbinding partner of the biological ligand being detected.

The present invention is based on the discovery that there is a class ofsuperparamagnetic particles which are small enough to function as tagsfor detection of biological ligands in ICT test formats where solidporous matrices are employed and also have a sufficiently large magneticmoment to function effectively as ligand concentration adjuvants. Thecapability of using the same particles for concentration and separationof a target ligand from a large volume of liquid and as tags for aqualitative ligand identification test or a similar test that not onlyidentifies but quantifies the amount of ligand enables a significantincrease in the sensitivity of the pre-assay concentration step. At thesame time, the separation of the target ligand from interfering orinhibitory substances that may be present in the original sample isenhanced, the awkward need for removing a magnetic label is avoided andso is the equally awkward need for introducing a second label.

BRIEF DESCRIPTION OF THE INVENTION

The present invention utilizes superparamagnetic subunits of 1-30 nm inaverage mean diameter, such as ferrofluid subparticles, which are mixedwith bovine serum albumin (“BSA”) or a similar biologically andmagnetically non-interfering substance capable of coating and coveringsuch particles, whereby they form BSA-coated ferrofluid particles whichspontaneously agglomerate to masses each containing a number offerrofluid subunit “cores” or nuggets, each completely surrounded byBSA. These BSA-ferrofluid agglomerates have been found to be highlyeffective, at overall particle average mean diameters of at least about100 nm ranging up to about 450 nm, and at times even higher, (1) asagents from concentrating and separating out target biological ligandsfrom large liquid volumes in which they are initially present in traceamounts and (2) as tags for enabling detection of these target ligands,for identity confirmation purposes and/or for quantification inICT-format assays.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 hereof is a plot of magnetic signal measured in millivolts (mV)against Respiratory Syncytial Virus (“RSV”) lysate in milligrams permilliliter at sample volumes of 0.1 ml., 1.0 ml. and 10.0 ml.,respectively.

DETAILED DESCRIPTION OF THE INVENTION

The vistas opened by the use of this invention are best appreciated froma consideration of the fact that the commercially available ICT assaysdescribed in the copending, commonly assigned U.S. patent applicationSer. No. 09/139,720 filed Aug. 25, 1998 and Ser. No. 09/397,110, nowU.S. Pat. No. 6,824,997 filed Sep. 16, 1999 as a continuation in part ofSer. No. 09/156,488 filed Sep. 18, 1998, now abandoned, which are bothhighly sensitive and specific for identifying the presence of particulardisease-causing bacteria, are successfully run with a few drops of testfluid—in the order of 100 microliters of urine, for example. Bacteriamolecules, however, are large in comparison to the molecules of, e.g.,viruses and various biochemical substances, the presence orconcentration of which may be indicative of a disease state or anotherabnormal condition in a human patient.

These smaller molecules often are widely dispersed in samples ofmammalian fluid, such as urine, with the result that the sample sizeadequate to enable detection of particular bacteria in the urine of aperson suffering from a disease of which those bacteria are causative,is too dilute to insure that smaller disease-causing molecules will beequally readily detectable.

By affording a means of concentrating the smaller molecules in a liquidsample prior to assaying for them, one is enabled to detect and, ifdesired, quantify, the presence in, e.g., human urine, of moleculesthat—if run in the assay format described in the aforementionedcopending applications, without preconcentration, could not be detectedwith high sensitivity and specificity and might not be detectable atall. Experience to date with pre-assay concentration usingsuperparamagnetic particles composed of ferrofluid 1-30 nm diametersubunits distributed in a BSA matrix, said composite particles havingaverage mean diameters of between at least about 100 nm and about 450 nmand coated with an antibody to the target molecule which attracts thetarget molecule and couples thereto, thereby effecting the desiredconcentration upon exposure to the gradient of a magnetic field, whenfollowed by an ICT assay for the target molecule which assay employs theaforesaid superparamagnetic particles as tags in the assay, hasdemonstrated a gain of approximately 2 logarithms of sensitivity to thetarget molecule over results heretofore attainable with methods whereinit was attempted to perform a conventional ICT assay for the targetmolecule on the original sample without concentration.

The superparamagnetic material used in the investigative work describedherein—i.e. ferrofluid core subunits of 1-30 nm diameter dispersed in amagnetically and biochemically inactive matrix of BSA—can be substitutedas to ferrofluid by any other metallic subunits of this size range thatexhibit superparamagnetic properties, including metals and metallicoxides which exhibit spinel structure alone or in combinations with oneanother. As already noted other materials that are magnetically inactiveand in themselves biochemically unreactive with the target ligand mayreadily be substituted for BSA.

The procedure for concentration of a target molecule in an aqueousmedium (including a mammalian bodily fluid such as urine, blood, saliva,sputum, etc.,) renders it necessary that the BSA-superparamagnetic coreagglomerates having a composite particle diameter of at least about 100mm be first coated with a material which is a binding partner for thetarget molecule. The coated superparamagnetic particles are thenimmersed in the fluid and incubated for a period of at least 15, andoften 30-40 or more, minutes. Complexes of superparamagnetic particlesand target ligand are thereby formed. These complexes are sequesteredfrom the bulk of liquid sample by exposure to the gradient of a magneticfield. The liquid is then removed by aspiration, decanting or any otherconvenient method and the particles are washed and dispersed in a volumeof a suitable buffer that is smaller than the volume of the originalsample. An ICT strip of nitrocellulose or other bibulous material uponwhich a stripe of binding partner for the target molecule—which may bethe same one used in the concentration step or a different one,depending upon the functionality of the target molecule—has beenimmovably bound to the capture zone area, contained in a “dipstick” ICTdevice format, is immersed in the buffered dispersion ofsuperparamagnetic particles complexes. Upon migration of these particlecomplexes along the strip, the target molecule on their outer surfacebinds to its binding partner in the immovable stripe, causingsuperparamagnetic particles to accumulate along the stripe. Experiencehas shown that immovable striping of binding partner for the targetmolecule in multiple lines, spaced apart from one another along the endof the strip remote from the sample receiving end, may be appropriate toensure efficient capture of the target ligand in this assay. Themagnetic signal of the superparamagnetic tag on the capture line orlines in millivolts, is read in a suitable instrument. The instrumentused for the work shown in the ensuing specific examples was a MagneticAssay Reader IV unit obtained from Quantum Design, Inc., San Diego,Calif.

This unit is especially designed to be compatible with small volumeassay formats, such as those which exhibit the end result as a line orlines of accumulated magnetic tag material. Because of the permeabilityof the magnetic field of the superparamagnetic tag, signal due to anyanalyte immobilized to the capture line is read as a single magneticmass. This is in contrast to readings obtained from optical inspectionwhich detect only the surface appearance of the capture line. Accordingto the manufacturer, the magnetic reading is linear with respect to themass of magnetic material on the capture line through at least fourorders of magnitude. The construction of standard curves correlatingmeasured magnetic signal to target ligand amount is readily achievableby methodology that is well known in the art.

It is anticipated that, for concentrating target molecules present inmammalian bodily fluids, such as, e.g. urine, saliva, blood, etc. atvery high dilution levels, it may at times be necessary to make use ofauxiliary magnetizable columns, filters or screens, or the addition ofnickel powder to the sample, to facilitate complete separation from thesample and from unbound particles, of the low mass of superparamagneticmaterial actually bound to target molecules.

The following examples, which are illustrative only and in no senselimiting, illustrate how the invention works in practice:

EXAMPLE 1

A partially purified viral lysate of respiratory syncytial virus (“RSV”)obtained from Chemicon (Catalog #Ag857, Lot 21031072) was diluted in anaqueous buffer of pH 7.8+0.1 having the following composition:

-   -   Tris base—24.22 grams per liter (g.p.l.)    -   Triton X-100—10 ml./liter    -   Tween 20—10 ml./liter    -   N-tetradecyl-N, N-dimethyl-3-ammonio-1-propane sulfonate—20.0        g.p.l.    -   Sodium azide—0.2 g.p.l.    -   Water added to make 1 liter        The resulting dilution contained 0.05 mg/ml. of RSV lysate.        Samples of 0.1 ml., 1 ml. and 10 ml., respectively of this        dilution were carefully withdrawn after thorough mixing. To each        of the 3 samples there was then added 5 microliters of        approximately 250 nm average mean diameter superparamagnetic        particles consisting of ferrofluid subunits of 1-30 nm diameter        embedded in and each separately surrounded by BSA, which        composite particles had been previously coated with anti-RSV        monoclonal antibody obtained from Viro Stat, Inc. (Catalog        #0631, Lot RM286). The mixture of the coated superparamagnetic        particles and buffered viral lysate was in each instance        thoroughly mixed and allowed to incubate for 30 minutes at room        temperature on a blood bag rotator platform. Each sample was        then exposed to the gradient magnetic field intensity produced        by a strong rare earth permanent magnet and held stationary for        at least 30 minutes, thereby concentrating the superparamagnetic        conjugate and any bound RSV lysate and sequestering them in the        area of greatest field intensity proximal to the magnet. In each        instance the supernatant was then removed by aspiration and 100        microliters of the above-described buffer was then added.

Each of the three resulting sample concentrates was thoroughly mixed andplaced in contact with a 22.5 mm wide nitrocellulose lateral flow ICTmembrane (purchased from Millipore Corp. and identified as HF07504, LotRK 000231) upon which had earlier been immovably striped across itswidth in the “capture” zone (located in the area most remote from thepoint of sample introduction) the same anti-RSV-monoclonal antibodyreferred to above. The contact with the nitrocellulose membrane, in eachinstance was initiated through an absorbent bridging pad, whereby themagnetic complex with analyte bound thereto was caused to migratelaterally through the strip to the capture line, along which magneticconjugate bound to the viral lysate analyte bound, but magneticconjugate free of viral lysate did not bind and was allowed to flow intoan absorbent zone positioned after the capture line. In each instancethe magnetic signal in millivolts of the capture line was read with theQuantum Design instrument referred to above. The exact procedure wasrepeated for each sample using run buffer alone, without the present ofRSV viral lysate, as a negative control. The measured results are shownin the following Table 1: TABLE #1 Measured Signal in mV RSV LysateConcentration 0.1 ml 1.0 ml 10 ml. in mg 1 ml sample sample sample 0 7.317.6 12.60 0.05 41.2 218.2 408.20

The results set forth in Table 1 are graphed in FIG. 1 hereof, whereinthe intensity of magnetically induced signal is represented as astraight line function of RSV lysate concentration in mg./ml. for eachsample volume assayed.

The signal to noise ratio and the detection limit in mg./ml. for eachsample were calculated and the results appear in the following Table 2:TABLE #2 RSV Lysate Concentration 0.05 mg. per ml. Signal to Noise RatioDetection Limit in mg./ml. 0.1 ml. sample 5.6 0.027 1.0 ml. sample 12.50.012 10 ml. sample 32.4 0.0046

Later work has shown that the total sensitivity of antigenic detectionfor each sample volume can be increased if multiple fixed stripes ofantibody are applied to the ICT membrane, each spaced apart from oneanother along the sample flow path and the total magnetic moment ofthese capture lines is measured.

This example as presented illustrates the efficacy and feasibility ofthe superparamagnetic particles of this invention, when coated with anappropriate biological ligand such as the antibody employed in the workunderlying this example, and thus enabling extraction of a biologicalligand from a dilute solution in which it occurs and thereby alsoconcentrating the ligand. It also illustrates the efficacy andpracticality involved in using the same superparamagnetic particles astags for the biological ligand in an ensuing ICT assay.

EXAMPLE 2

This example involves a possible use of the superparamagnetic particlesdescribed herein in an experimental ICT test for quantifying Legionellapneumophila serogroup 1 in environmental water. The present commerciallyavailable test is described in copending, commonly assigned U.S.application Ser. No. 09/458,998 filed Dec. 10, 1999 as acontinuation-in-part of copending, commonly assigned U.S. applicationSer. No. 09/139,770 filed Aug. 25, 1998.

In this experiment, two 100 ml. samples of cooling tower water weredrawn at Hood Dairy, Portland, Me. and held at temperature of 2-8° C. Toone of these samples was added sufficient Legionella pneumophilaserogroup 1 bacteria to enrich the sample bacteria content by 95colony-forming units (“CFU”) per ml. Both samples were subjected to afiltration concentration on a small pore membrane as described in detailin copending U.S. application Ser. No. 09/458,998. The particulateretained on the membrane was recovered on a swab in each instance andwas reconstituted to a sample volume of 200 μl with a buffer compositioncomposed of aqueous 0.05 M Tris HCl and 2.5% Tween 20 having a pH of7.0±0.1. Each sample was then carefully split into two portions. Allfour resulting samples were of equal volume.

Particles of approximately 100 nm average mean diameter composed offerrofluid subunits, each of 1-30 nm average mean diameter, distributedin an enveloping matrix of BSA were coated with anti-Legionellapneumophila serogroup 1 antibodies which had been purified as describedin copending U.S. application Ser. No. 09/139,720. Equal amounts of theresulting conjugate were added to all four of the samples and allowed toincubate for 15 minutes. Each sample was then exposed to the gradient ofa strong magnetic field as in Example 1. One sample having no addedLegionella pneumophila serogroup 1 bacteria and one sample having 95CFu/ml of added bacteria were immediately subjected to an ICT testperformed with a dipstick style lateral flow device comprising anitrocellulose membrane pretreated by the application of a fixed stripeof purified anti-Legionella pneumophila serogroup 1 antibodies at theend of the strip most remote from the point of sample-introduction. Thetest strips and sample in each instance were not washed; once thesamples had migrated to the end of the nitrocellulose membrane, theconductivities in millivolts (mV) of the capture lines were read by theQuantum Design instrument referred to above. The sample having no addedbacteria gave a negative reading of −248.4 mV, which is believedattributable to the presence in the sample of particulate that was notbroken down. The sample with added bacteria (95 CFU/ml) gave a readingof 375 mV.

The remaining two samples were each washed 3 times with 500 ml. of thebuffer and then reconstituted to 100 μl and run in the same manner in anidentical ICT test, to that of the first two samples. The magneticmoments of the capture lines of each of the respective test strips wereread in the Quantum Design instrument. The washed sample having no addedbacteria gave a reading of 8.5 mV. The washed sample with added bacteriagave a reading of 161.8 mV.

The example supports the broad concept of superparamagnetic alloyseparating the target ligand—in this instance the O-polysaccharideantigen of Legionella pneumophila serogroup 1—from a liquid sample,using superparamagnetic particles, followed by conducting an immunoassayusing the same supermagnetic label for detection.

Those skilled in the art will recognize many opportunities for makinguse of the particles and methods referred to herein beyond thepossibilities explicitly disclosed. It is therefore intended that thescope of this invention be limited only by the appended claims.

1. A process for (1) separating a target biological ligand suspected ofbeing present in dilute concentration in an aqueous fluid and (2)ascertaining whether said target ligand is present, which processcomprises the steps of a) coating with a first biological bindingpartner for said target biological ligand a group of superparamagneticparticles, which particles have an average mean diameter of at leastabout 100 nm and are each composed of superparamagnetic subunits, whichsubunits have an average mean diameter of 1-30 nm and are separatelyspaced from one another within a covering matrix of nonmetallic,non-magnetic material that is compatible, but non reactive, with saidbiological ligand and its first biological binding partner, b) immersingthe coated superparamagnetic particles from step (a) in a sample of saidaqueous fluid which is suspected of containing said target biologicalligand and allowing said particles and said fluid to incubate for a timesufficient to allow the target biological ligand, if present, to reactwith its first biological binding partner coated on said particles,thereby forming complexes, c) exposing said particles to the gradient ofa magnetic field, whereby said they acquire a magnetic charge and areattracted to one another, d) removing said particles from said fluidwith a permanent magnet, e) washing said particles f) releasing saidmagnetic field and removing said particles, g) adding to said particlesa small volume of an aqueous buffer to form a dispersion of saidparticles in said buffer h) applying said dispersion from step (g) tothe sample receiving end of an immunochromatographic (“ICT”) devicecomprising a strip of bibulous material having at least one imrnmoveablestripe of a second binding partner for said ligand affixed permanentlythereto at a position near the end of said strip that is opposite itssample receiving end, (i) allowing said dispersion to migrate along saidstrip and contact said at least one immovable stripe of a second bindingpartner for said biological ligand, whereby target biological ligand onthe surface of said particles binds to its second binding partner onsaid immovable stripe, and (j) observing whether a mass of the metallicmaterial from said particles, indicative of the presence of said targetligand in said sample, appears along said immovable stripe.